Frederic Vigant¹, Jihye Lee², Axel Hollmann³, Lukas B. Tanner^4,5, Zeynep Akyol Ataman¹, Tatyana Yun⁶, Guanghou Shui^7¤a, Hector C. Aguilar⁸, Dong Zhang⁹, David Meriwether¹⁰, Gleyder Roman-Sosa^11¤b, Lindsey R. Robinson¹, Terry L. Juelich⁶, Hubert Buczkowski¹², Sunwen Chou¹³, Miguel A. R. B. Castanho³, Mike C. Wolf^1¤c, Jennifer K. Smith⁶, Ashley Banyard¹², Margaret Kielian¹¹, Srinivasa Reddy¹⁰, Markus R. Wenk^4,14,15, Matthias Selke⁹, Nuno C. Santos³, Alexander N. Freiberg⁶, Michael E. Jung², Benhur Lee^1*

¹ Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America, ² Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America, ³ Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal, ⁴ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, ⁵ NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, Singapore, ⁶ Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America, ⁷ Life Sciences Institute, National University of Singapore, Singapore, ⁸ Paul G. Allen School for Global Animal Health, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America, ⁹ Department of Chemistry and Biochemistry, California State University, Los Angeles, California, United States of America, ¹⁰ Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, ¹¹ Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America, ¹² Wildlife Zoonoses and Vector Borne Disease Research Group, Animal Health and Veterinary Laboratories Agency, Weybridge, Surrey, United Kingdom, ¹³ Oregon Health & Science University and VA Medical Center, Portland, Oregon, United States of America, ¹⁴ Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, ¹⁵ Swiss Tropical and Public Health Institute and University of Basel, Basel, Switzerland

Abstract
LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC₅₀#0.5 mM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001’s specific inhibition of viruscell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (¹O₂) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated ¹O₂. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. ¹O₂-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced ¹O₂-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001’s mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001’s limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC₅₀,10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of ¹O₂ generation), and (4) 10.100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p#0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.

¤a Current address: State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

¤b Current address: Department of Internal Medicine I, University of Ulm, Ulm, Germany.

¤c Current address: Defense Threat Reduction Agency, Fort Belvoir, Virginia, United States of America.